α-Aryl-N-aryl nitrones: Synthesis and screening of a new scaffold for cellular protection against an oxidative toxic stimulus

Article abstract of DOI:10.1016/j.bmc.2015.12.007

Nitrone-containing compounds are commonly employed as spin traps of free radical species in chemical and biological studies. Some molecules as α-phenyl-N-t-butyl nitrone (PBN) and its derivatives have been tested as potential drugs to treat oxidative stress related diseases, as Alzheimer and stroke for example. In this work we report the design and the synthesis of α-aryl-N-aryl nitrones and their cytoprotection profile on human neuroblastoma cells (SH-SY5Y) under induced oxidative stress. All the nine synthesized nitrones showed a significant response at low micromolar concentration. The selected compound 8 (α-phenyl-N-phenyl nitrone) increased the reduced glutathione (GSH) levels by 65% and lowered the necrotic cell death from 25.8% to 3.8%. Based on our data, the designed highly conjugated nitrone double-bond skeleton can be considered as a good scaffold for further studies regarding oxidative stress-related diseases.

Full text of DOI:10.1016/j.bmc.2015.12.007

Bioorganic & Medicinal Chemistry 24 (2016) 232–239
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Bioorganic & Medicinal Chemistry
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a
-Aryl-N-aryl nitrones: Synthesis and screening of a new scaffold for
cellular protection against an oxidative toxic stimulus
y
y
à
à,
⇑
A. C. Matias , G. Biazolla , G. Cerchiaro , A. F. Keppler
Center for Natural Science and Humanities, Federal University of ABC—CCNH, UFABC, Avenida dos Estados, 5001, S610-3-Tower A, 09210-580 Santo Andre, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitrone-containing compounds are commonly employed as spin traps of free radical species in chemical
Received 15 October 2015
Revised 27 November 2015
Accepted 5 December 2015
Available online 10 December 2015
and biological studies. Some molecules as
tested as potential drugs to treat oxidative stress related diseases, as Alzheimer and stroke for example. In
this work we report the design and the synthesis of -aryl-N-aryl nitrones and their cytoprotection pro-
a-phenyl-N-t-butyl nitrone (PBN) and its derivatives have been
a
file on human neuroblastoma cells (SH-SY5Y) under induced oxidative stress. All the nine synthesized
nitrones showed a significant response at low micromolar concentration. The selected compound 8
(a-phenyl-N-phenyl nitrone) increased the reduced glutathione (GSH) levels by 65% and lowered the
necrotic cell death from 25.8% to 3.8%. Based on our data, the designed highly conjugated nitrone
double-bond skeleton can be considered as a good scaffold for further studies regarding oxidative
stress-related diseases.
Keywords:
Synthesis
Nitrone
Cytoprotection
Oxidative stress
SH-SY5Y
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
ii can undergo oxidative degradation to benzaldehyde and N-
2
1
oxide, which is subsequently decomposed to NO; therefore,
PBN should act as an NO cell signaling source (Scheme 1B). More
recently, Das et al. proposed that the cytoprotection by nitrones
comprises a combination of ROS quenching, phase II enzyme
upregulation and mitochondria-mediated apoptotic cascade sup-
Under certain circumstances, the levels of radical species (RS)
rise beyond the natural metabolic capacity of the body. The elimi-
nation of radical oxygen species (ROS) and radical nitrogen species
(
RNS) occurs predominantly through an intricate defense system in
2
5
which enzymes and nonenzymatic antioxidants function to protect
the organism. The imbalance between ROS generation and elimina-
tion leads to deleterious effects called oxidative stress, causing
damage to proteins, lipids and nucleic acids and resulting in the
impairment of vital functions of cells, tissues and organs. Alzhei-
pression (Scheme 1C). Taking into account the above mentioned
mechanisms, it remains unclear whether the antioxidant responses
are due to the interaction of nitrones or nitrones-ROS adducts with
biomolecules or whether the observed effects are due to other indi-
rect mechanisms.
mer’s disease, atherosclerosis,3 cancer,
1
,2
4–6
diabetes,7 inflamma-
obesity,10–12 Parkinson’s disease13–15 are pathologies
related to oxidative stress, which can be observed early or at
In this work, we report the synthesis and cytoprotective profile
8
,9
tion,
of a panel of nitrones with a nitrone-included
as a common fragment in their structure (Fig. 1). Because the most
common substituents on an -N-nitrone moiety are benzyl and
alkyl groups, mainly t-butyl and methyl,
p-conjugated moiety
1
6–18
advanced stages.
For years, nitrone-bearing molecules such as
a
2
5–31
a
-phenyl-N-t-
we selected aryl
butyl nitrone (compound i, Scheme 1) have been tested as poten-
groups as N-nitrone substituents, focusing on two different unex-
plored aspects in the field of bioactive nitrone-bearing molecules:
(a) the development of a highly restricted rotation skeleton and (b)
the two-way conjugation of double bonds covering the nitrone
moiety (Fig. 1).
tial drug candidates to treat oxidative stress-related diseases,
many of them presenting interesting results.1
9–24
Mechanisms of
action have been proposed to explain the in vitro and in vivo
antioxidant profiles of nitrones, wit direct ROS scavenging
(
Scheme 1A). Chamulitrat et al. showed that PBN-radical adduct
It has been reported that the antioxidant property of nitrones
could be improved by extending the double-bond conjugation.32
This electronic effect is also observed in other classes of mole-
⇑
Corresponding author. Tel.: +55 11 4996 8358 (office), +55 11 4996 7458 (lab).
E-mail address: artur.keppler@ufabc.edu.br (A.F. Keppler).
Contributed equally to the development of this work.
3
3–36
cules.
On our proposed scaffold (Fig. 1), the highly conjugated
y
framework restricts rotation in the planar structure, maintaining
the nitrone moiety in extended two-way conjugation. Comparing
à
Contributed equally to the intellectual planning of this work and discussion of the
present manuscript.
http://dx.doi.org/10.1016/j.bmc.2015.12.007
0
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

A. C. Matias et al. / Bioorg. Med. Chem. 24 (2016) 232–239
233
Scheme 1. Proposed antioxidant/cytoprotective mechanisms of action of
a-phenyl-N-t-butyl nitrone against oxidative stress.
2
2
. Results and discussion
.1. Molecular structure planning and synthetic chemistry
Based on valuable seminal reports and to contribute to the
understanding of the cytoprotective and antioxidant properties of
the nitrone functional group, we proposed to synthesize a library
of novel nitrones with extended conjugation for biological pro-
poses. To this end, we synthesized a panel of
a-aryl-N-aryl nitrones
with moderate to good yields (Scheme 2) by reacting commercial
3
7
aryl aldehydes with nitroarenes of interest. The use of aryl rings
bearing the nitrone group offered the opportunity to assess a vast
library of commercial starting materials, potentially opening the
door for a new generation of nitrone-containing antioxidant
compounds.
The steric effects of aryl substituents on
a-aryl-N-aryl nitrones
dominate the reaction’s stereospecificity, resulting in the most
2
7,38
stable Z isomer.
All synthesized compounds were assigned as
the Z-configuration by comparing NMR data with those reported
for known nitrones and presented analytical and spectral data in
accordance with their structures (see Section 4).
Figure 1. Representation of the two-way conjugation of
a-aryl-N-aryl nitrones, the
restricted conjugation of -alkyl or -benzyl-N-aryl nitrones and a retrosynthetic
a
a
proposal to obtain two electronically distinct classes of nitrones.
In addition, we were also interested in analyzing the relation-
ship between structure and the cytoprotective capacity of the
a-
aryl-N-aryl nitrone class. Thus, compounds 1, 2, and 3 were
designed with two electron-donating methyl groups on the N-aryl
ring; 4, 5, and 6 bear an electron-withdrawing chloride; followed
by 7, 8, and 9, without any substituent on the N-aromatic ring.
to other classes of nitrones, the conjugation enhancement on
a-
aryl-N-aryl nitrones would assist in the understanding of the struc-
ture–cytoprotective activity relationship of nitrone-containing
molecules.
For the
a-aryl substituent of nitrone moiety, the compounds 1, 4,
For comparison of the cytoprotective profile of the synthesized
-aryl-N-aryl nitrones, an in vitro MTT assay using human neurob-
and 7 have a pyridine as an aryl group; 2, 5, and 8 have no sub-
stituent; and 3, 6, and 9 bear a 3,4-methylenedioxy donating
group. Although the syntheses of 1, 2, 5, 7, 8 and 9 have been
a
lastoma cells (SH-SY5Y) under H toxic stimulation was per-
2 2
O
3
9–43
formed. To analyze the cytoprotective role of nitrones at the
cellular level, in further studies, we examined the intracellular
levels of total and reduced glutathione, SOD activity and cell death.
Based on the collected data, we suggest that cellular pretreat-
ment with nitrone compounds modulates the levels of natural
antioxidants, increasing the intracellular concentrations of reduced
reported previously,
no biological activity has been described
for these compounds; compounds 3, 4 and 6 are novel in the
literature.
2.2. In vitro evaluation of the cytoprotective and cytotoxicity
profiles of nitrone derivatives
glutathione (GSH). Among the screened novel compounds,
a-(Z)-
phenyl-N-phenyl-nitrone (compound 8, Scheme 2) exhibited the
highest cytoprotective performance, with 20% recovery of cell via-
bility and ꢀ6.0% less necrotic death in comparison to its N-alkyl
analog.
2 2
The cytoprotective profile of nitrones 1–9 against H O toxic
stimuli was evaluated by the MTT metabolic reduction assay using
the human neuroblastoma SH-SY5Y cell line, a well-known in vitro
1
,22,44
model for neuronal studies.
By incubating SH-SY5Y cultures

2
34
A. C. Matias et al. / Bioorg. Med. Chem. 24 (2016) 232–239
Scheme 2. Synthetic strategy and yields of synthesized a-aryl-N-aryl nitrones.
for 48 h with different concentrations of H
2
O
2
, we identified
most potent molecule of the panel, with 42% and 95% better results
compared to the positive and negative controls, respectively. Inde-
pendent of the substituent on both aromatic groups, all of the syn-
3
00 M as the optimal toxic condition (Fig. 2). Using this experi-
l
mental condition, a substantial increase in cell viability was
observed by pretreating the SH-SY5Y cultures with nitrones at con-
thesized nitrones presented cytoprotection against the H
oxidative stress.
2 2
O
centrations of 1
Phenyl-N-t-butyl nitrone (PBN) was used as a positive control. At
M, the lowest concentration studied, all synthesized nitrones
lM (Fig. 3A), 10 lM and 100 lM (Fig. 3B and C). a-
1
l
2.3. Cell death by flow cytometry analysis
resulted in an increase in cell viability of 71% compared to cells
with no nitrone pretreatment and an increase of 42% compared
to cells pretreated with PBN (Fig. 3). Nitrone 2 proved to be the
To clarify the effect of the decrease in cell viability observed
when cells without nitrone pretreatment were exposed to H
negative control), we performed flow cytometry analyses with
Annexin-V/FITC and PI labeling. Cells were pretreated with PBN
and nitrones 2 and 8 for 24 h and then treated with 300 M H
2 2
O
(
l
2 2
O .
A positive control for cell death was performed with 100 nM stau-
rosporine. The results of the analyses (Fig. 4) showed that SH-
SY5Y cells exhibited 96.33 ± 1.4% viability, with 1.37 ± 0.67% being
in the late stage of apoptosis and 0.13 ± 0.96% being necrotic. In
contrast, the negative control showed a 26% reduction in viability
(
(
71.08 ± 2.34%) and an increasing number of necrotic cells
25.81% ± 3.57) and late apoptosis (2.78% ± 0.53%), confirming that
the decreased cell viability is related to necrosis. Independently of
the compound structure, the cells pretreated with nitrones showed
8
5–95% viability, and 3.8–9.4% were necrotic. These results suggest
that both PBN and the synthesized nitrones inhibit the cytotoxic
effects of H in neuroblastoma cells. Nitrone 8 exhibited the
2 2
O
greatest cytoprotective effect because the percentage of necrotic
cells was decreased by ꢀ22% and 6% relative to the negative and
positive controls, respectively.
2
.4. Effects of nitrones in the generation of reactive oxygen
species (ROS)
Figure 2. Effect of H
with increasing concentrations of H
2
O
2
per se on SH-SY5Y cells. The cells were incubated for 48 h
(50–500 M). The data measured by MTT
2 2
O
0
0
assay were correlated as an index of cell proliferation. Data represent the mean
Using the oxidant-sensing probe 2 ,7 -dichlorodihydrofluores-
cein diacetate (DCFH-DA) and flow cytometry, we assessed oxida-
values ± standard deviation (n = 5), significant differences between treated and
⁄
untreated cells were p < 0.005 ( ) and significant differences between treated and
⁄
⁄
2 2
tive stress in SH-SY5Y cells during H O toxic stimulus. We
untreated cells were p < 0.001 ( ).

A. C. Matias et al. / Bioorg. Med. Chem. 24 (2016) 232–239
235
Figure 3. (A) In vitro evaluation of nitrone cytoprotective profiles at 1
control, Cꢁ) or pretreated for 24 h with PBN (positive control, C+) or with different nitrones at 1, 10 and 100
stimulus. Data measured by the MTT assay were correlated as an index of cell proliferation. The data represent the mean values ± standard deviation (n = 5); significant
l
M in SH-SY5Y cells treated with H
2
O
2
, (B) at 10
lM and (C) at 100
l
M. Cells were untreated (negative
2 2
lM and then exposed to H O (300 lM) for 24 h as a toxic
⁄
⁄
differences between treated and untreated cells (negative control, Cꢁ) were p<0.001 ( ), and significant differences between treated cells and positive control (PBN, C+) were
p<0.001 (##).
Figure 4. Apoptosis analysis by flow cytometry. SH-SY5Y cells were treated with 300
and analyzed by flow cytometry with Annexin-V/FITC and PI staining. Cells untreated and treated with 100 nM staurosporine were used as controls of viable and dead cells,
l
M H
2
O
2
(negative control, Cꢁ), PBN (positive control, C+), or nitrones 2 and 8 (1 lM)
respectively. Data represent the mean values ± standard deviation (n = 3).

2
36
A. C. Matias et al. / Bioorg. Med. Chem. 24 (2016) 232–239
Figure 5. Effects of nitrones on oxidative stress. Dot plots of intracellular DCF-DA fluorescence in SH-SY5Y cells pretreated with 2 and 8 for 24 h and subsequently treated
with 300 M H
for 24 h. Data represent the mean values ± standard deviation (n = 5); significant differences between treated and untreated cells (negative control, Cꢁ)
were p <0.001 ( ).
l
2 2
O
⁄
⁄
observed that cells pretreated with 2, 8 and PBN had a lower fluo-
3. Conclusions
rescent response in comparison to cells without nitrone pretreat-
ment (Fig. 5). Our results suggest that pretreatment with
nitrones can inhibit oxidative stress.
Considering the above results, we conclude that 24 h of pre-
treatment with
a
-aryl-N-aryl nitrones protects SH-SY5Y cells from
. Correlating the MTT data with the cell
the toxic effects of H
2 2
O
2
.5. Effect of nitrones on natural antioxidants levels
proliferation index, all of the synthesized nitrones presented simi-
lar or better cytoprotective effects than PBN, the nitrone compound
To gain further insight into the cellular pathway and cytopro-
of reference. The flow cytometry obtained data showed that the a-
aryl-N-aryl nitrones 2 and 8 significantly reduced necrosis caused
tective mechanism of the synthesized
a-aryl-N-aryl nitrones dur-
ing H toxic stimulus, we investigated the intracellular ratio of
2
O
2
by H O , from 28.5% necrotic cells to 11.2–5.2%. The use of
2
2
reduced and total glutathione.
DCFH-DA fluorescent sensing probe indicated that nitrones 2 and
8 depleted the oxidative stress and by GSH quantification, we
observed the increase of reduced glutathione (GSH) levels by
65%. Based upon these results we propose that the two-way conju-
Using experimental conditions similar to those employed for
quantifying cell viability, we observed that pretreatment with
nitrones had no effect on total levels of glutathione in SH-SY5Y
cells (Fig. 6A) but significantly increased the level of reduced glu-
tathione (Fig. 6B). Isolated, this result may indicate an upregulation
gated skeleton of
a-aryl-N-aryl nitrones is a good scaffold for the
development of new drugs to more efficiently reduce the deleteri-
ous effects associated with a state of oxidative stress.
of GPx, as proposed recently.25 Conversely, the H
2 2
O toxic stimulus
appeared to generate a significant redox imbalance favoring the
reduced substrate, which was measured by the high concentration
of reduced glutathione (Fig. 6B).
Pretreatment with nitrones 2, 8 and PBN increased GSH levels
by 65% compared to without nitrone pretreatment (Cꢁ, Fig. 6B).
Herein, our data suggest that the studied nitrone-containing com-
pounds act as a trigger, resulting in an increase of the GSH rate of
production, greater than its rate of consumption.
4
4
. Experimental section
.1. General methods
Reactions were monitored by TLC using precoated silica gel alu-
minum plates containing a fluorescent indicator (Merck, 5539).
Product and starting material detection was performed by UV light
Figure 6. In vitro evaluation of the effect of nitrones on natural GSH antioxidant levels in SH-SY5Y cells stressed with H
levels, A and B, respectively. Cells were untreated (negative control, Cꢁ) or pretreated for 24 h with PBN (positive control, C+) or with 2 and 8 (1
(300 M) toxic stimulus for 24 h. Data represent the mean values ± standard deviation (n = 5); significant differences between treated and untreated cells (negative
control, Cꢁ) were p <0.001 ( ).
2
O
2
, as expressed by the quantification of GSHx or GSH
l
M) and then exposed to
H
2
O
2
l
⁄⁄

A. C. Matias et al. / Bioorg. Med. Chem. 24 (2016) 232–239
237
absorption at 254 nm. Anhydrous MgSO
solutions during work-ups, and the removal of solvents was carried
out under vacuum with rotary evaporator. Flash column
chromatography was performed using Silica Gel Flash 60
4
was used to dry organic
126.4, 125.1, 122.6, 118.6, 108.6, 108.4, 101.6, 19.9, 19.5. HRMS
(EI) m/z calcd for C16 N 270.1130, found 270.1120.
16 3
H O
a
4.2.4. a-(Z)-4-Pyridyl-N-4-chlorophenyl nitrone (4)
(
0.035–0.075, pore diameter ꢀ60 nm, Aldrich). Melting points
Following the general procedure at a 10 mmol 4-pyridinecar-
boxaldehyde scale, compound 4 was purified by column chro-
were determined using a Kofler block and are uncorrected. IR
spectra were obtained with a Perkin–Elmer Spectrum One spec-
trophotometer using an ATR accessory. One-dimensional H NMR
and C NMR spectra were recorded at 298 K with a Bruker Avance
III spectrometer operating at a frequency of 300.13 MHz and
matography and isolated as
a
white powder (1.39 g, 60%):
133–135 °C. H NMR (300 MHz, CDCl ): d 8.77 (d, 2H, J = 3 Hz),
8.15 (d, 2H, J = 3 Hz), 7.94 (s, 1H), 7.74 (m, 2H), 7.49 (m, 2H).
NMR (75 MHz, CDCl ): d 150.7, 147.2, 136.7, 132.3, 129.5, 123.0,
1
1
3
1
3
13
C
3
7
5.00 MHz, respectively. Chemical shifts are reported in parts per
2
121.5. HRMS (EI) m/z calcd for C12H10ON Cl 233.0482, found
million (ppm), and tetramethylsilane was used as an internal stan-
dard. High-resolution mass spectrometry spectra were obtained
using a Carlo Erba EA 1108 apparatus. All reagents and solvents
were purchased from Sigma Chemical Co. and were used without
purification.
233.0476.
4.2.5. a-(Z)-Phenyl-N-4-chlorophenyl nitrone (5)
Following the general procedure at a 10 mmol benzaldehyde
scale, compound 5 was purified by column chromatography (hex-
ane/EtOAc from 5:1 to 2:1, v/v) and isolated as a light yellow pow-
1
4
.2. General procedure for the synthesis of
a-aryl-N-aryl
der (1.73 g, 75%): mp 175–181 °C NMR H (300 MHz, CDCl
3
): d
nitrones
8.42–8.39 (m, 2H), 7.92 (s, 1H), 7.79–7.74 (m, 2H), 7.53–7.46
1
3
(
m, 4H). NMR C (75 MHz, CDCl
130.4, 129.3, 129.1, 128.7, 123.0. HRMS (EI) m/z calcd for
11ONCl 232.0529, found 232.0530.
3
): d 147.5, 135.8, 134.5, 131.2,
The compounds were prepared using a procedure described in
2
9
the literature, with slight modifications. A nitroarene (2 equiv)
and elemental zinc (3 equiv) were added to a three-neck round-
bottom flask containing an ethanolic solution of the appropriate
aldehyde (0.14 M). Next, this suspension was cooled to 0 °C, and
glacial acetic acid (6 equiv) was added slowly with mechanical stir-
ring. The solution was allowed to reach room temperature and was
stirred for an additional 2 h. The precipitate was filtered and
washed with EtOAc (3 ꢂ 20 mL). The combined organic layers were
13
C H
4.2.6.
nitrone (6)
Following the general procedure at a 10 mmol 3,4-(methylene-
a-(Z)-3,4-(Methylenedioxy)phenyl-N-4-chlorophenyl
dioxy)benzaldehyde scale, compound 6 was purified by column
chromatography (hexane/EtOAc from 5:1 to 2:1, v/v) and isolated
1
as a yellow powder (1.92 g, 70%): mp 155–163 °C NMR
(300 MHz, CDCl
H
dried over MgSO
4
and filtered, and the solvent was concentrated by
3
): d 8.29 (d, 1H, J = 3 Hz), 7.82 (s, 1H), 7.72 (dt,
rotary evaporation. The resulting solid was purified by flash
column chromatography (hexane/EtOAc from 5:1 to 2:1, v/v) to
generate pure compounds.
2H, J = 5 and 1 Hz), 7.69 (dd, 1H, J = 5 and 1 Hz), 7.44 (dd, 2H,
J = 6 and 3 Hz), 6.91 (d, 1H, J = 6 Hz), 6.91 (d, 1H, J = 3 Hz), 6.06
1
3
(s, 2H). NMR C (75 MHz, CDCl
34.1, 129.1, 125.4, 125.3, 122.8, 108.5, 101.6. HRMS (EI) m/z calcd
for C14 NCl 276.0428, found 276.0416.
3
): d 149.9, 147.7, 147.2, 135.4,
1
4
.2.1.
a-(Z)-4-Pyridyl-N-3,4-dimethylphenyl nitrone (1)
11 3
H O
Following the general procedure at a 10 mmol 4-pyridinecar-
boxaldehyde scale, compound 1 was purified by column chro-
4.2.7.
Following the general procedure at a 10 mmol 4-pyridinecar-
boxaldehyde scale, compound was purified by column
chromatography and isolated as a white crystal (1.62, 72%): mp
a-(Z)-4-Pyridyl-N-phenyl nitrone (7)
matography and isolated as a white powder (1.34 g, 68%): mp
1
1
10–116 °C. NMR H (300 MHz, CDCl
3
): d 8.74 (d, 2H, J = 6 Hz),
7
8
.16 (d, 2H, J = 6 Hz), 7.93 (s, 1H), 7.55 (s, 1H), 7.46 (d, 1H,
13
1
J = 3 Hz), 7.24 (d, 1H, J = 3 Hz), 2.34 (s, 6H). NMR C (75 MHz,
CDCl ): d 150.5, 146.8, 139.8, 138.0, 137, 131.8, 130.1, 122.6,
3
137–141 °C. H NMR (300 MHz, CDCl ): d 8.76 (dd, 2H, J = 4.4 and
3
1.5 Hz), 8.17 (dd, 2H, J = 4.4 and 1.5 Hz), 7.97 (s, 1H), 7.78 (m,
1
3
1
21.5, 118.8, 19.9, 19.6. HRMS (EI) m/z calcd for C15
H16ON
2H), 7.53 (m, 3H). C NMR (75 MHz, CDCl
3
): d 150.7, 147.2,
2
27.1185, found 227.1178.
136.7, 132.3, 130.0, 129.5, 123.0, 121.6. HRMS (EI) m/z calcd for
12 2
C H11ON 199.0872, found 199.0894.
4
.2.2.
a-(Z)-Phenyl-N-3,4-dimethylphenyl nitrone (2)
Following the general procedure at a 10 mmol benzaldehyde
scale, compound 2 was purified by column chromatography and
4
.2.8.
a-(Z)-Phenyl-N-phenyl nitrone (8)
Following the general procedure at a 10 mmol benzaldehyde
isolated as a light yellow powder (1.45 g, 74%): mp 58–66 °C
scale, compound 8 was purified by column chromatography (hex-
ane/EtOAc from 5:1 to 2:1, v/v) and isolated as a light yellow pow-
der (1.75 g, 78%): mp 107–111 °C NMR H (300 MHz, CDCl
1
NMR H (300 MHz, CDCl
3
): d 8.39–8.36 (m, 2H), 7.87 (s, 1H), 7.55
1
3
(
(
1
C
d, 1H), 7.46–7.40 (m, 4H), 7.15 (d, 1H), 2.28 (s, 6H). NMR
75 MHz, CDCl ): d 146.6, 138.4, 137.4, 133.7, 130.6, 130.4, 129.7,
28.7, 128.2, 122.3, 118.4, 19.6, 19.2. HRMS (EI) m/z calcd for
16ON 226.1232, found 226.1223.
C
1
3
): d
3
8
6
1
C
.43–8.40 (m, 2H), 7.93 (s, 1H), 7.79–7.76 (m, 2H), 7.48–7.45 (m,
13
H). NMR C (75 MHz, CDCl
29.0, 128.5, 125.4, 122.2, 121.6. HRMS (EI) m/z calcd for
12ON 198.0919, found 198.0908.
3
): d 148.9, 134.4, 130.8, 129.8,
15
H
13
H
4
.2.3. a-(Z)-3,4-(Methylenedioxy)phenyl-N-3,4-dimethylphenyl
nitrone (3)
4.2.9. a-(Z)-3,4-(Methylenedioxy)phenyl-N-phenyl nitrone (9)
Following the general procedure at a 10 mmol 3,4-(methylene-
Following the general procedure at a 10 mmol 3,4-(methylene-
dioxy)benzaldehyde scale, compound 3 was purified by column
dioxy)benzaldehyde scale, compound 9 was purified by column
chromatography (hexane/EtOAc from 5:1 to 2:1, v/v) and isolated
chromatography (hexane/EtOAc from 5:1 to 2:1, v/v) and isolated
1
1
as a light yellow powder (1.83 g, 76%): mp 125–129 °C NMR
300 MHz, CDCl
J = 6 Hz), 7.56 (s, 1H), 7.44 (d, 1H, J = 9 Hz), 7.19 (d, 1H, J = 9 Hz),
H
as a yellow crystal solid (2.20 g, 82%): mp 130–134 °C NMR
(300 MHz, CDCl
J = 6 Hz), 7.70 (d, 1H, J = 3 Hz), 7.46–7.44 (m, 3H), 6.89 (d, 1H,
H
(
3
): d 8.31 (s, 1H), 7.80 (s, 1H), 7.68 (d, 1H,
3
): d 8.32 (s, 1H), 7.83 (s, 1H), 7.74 (d, 2H,
1
3
13
6
.90 (d, 1H, J = 9 Hz), 6.04 (s, 2H), 2.32 (d, 1H, J = 6 Hz), NMR
C
J = 6 Hz), 6.03 (s, 2H). NMR C (75 MHz, CDCl
3
): d 149.6, 148.7,
(
75 MHz, CDCl ): d 149.5, 147.6, 146.9, 138.5, 137.7, 133.7, 130.0,
3
147.6, 134.1, 129.6, 129.0, 125.2, 125.1, 121.5, 108.5, 108.4,

2
38
A. C. Matias et al. / Bioorg. Med. Chem. 24 (2016) 232–239
cytometry at 530 nm47 using a Cytometer BD FACSCanto II.48,49
Assays were performed at least in triplicate, and >20,000 viable
cells from each sample were analyzed per assay.
TM
1
2
12 3
01.6. HRMS (EI) m/z calcd for C14H O N 242.0817, found
42.0812.
4
.3. Cell culture
4
.7. Determination of the GSH/GSSG ratio
Cell media were prepared with DNase- and RNase-free water
and filtered through 0.22- m filter membranes (Millex GV, Milli-
pore) prior to use. The cell cultures were manipulated using sterile,
disposable nonpyrogenic plasticware and were maintained at 37 °C
2
in an atmosphere of 5% CO in air at a relative humidity of 80%.
Human neuroblastoma SH-SY5Y cells were purchased from
American Type Culture Collection (ATCC) and grown in Dulbecco’s
Modified Eagle F12 Medium (DMEM/F12) supplemented with 10%
heat-inactivated fetal bovine serum (Gibco), 100 U/ml penicillin
l
SH-SY5Y cells were plated and preincubated with PBN, 2 and 8
all at concentrations of 1.0
with 300 M H for 24 h. The cells were trypsinized, washed
three times with cold PBS and centrifuged (1500g, 3 min, 4 °C).
The cells were then resuspended in 200 l of cold water and lysed
by freezing in a mixture of dry ice and ethanol for 10 min. After
thawing, the proteins were precipitated with 50 l of 10% sulfosal-
icylic acid and centrifuged (4000g, 5 min, 4 °C). The protein con-
centration in the pellet was determined, and the levels of GSH
and GSSG in the supernatant were assessed using a previous proto-
lM, for 24 h, and subsequently treated
l
2 2
O
l
l
and 10.0 lg/ml streptomycin. The cells were routinely trypsinized
4
2
and seeded at a density of 4 ꢂ 10 cells/cm . Every month, the cells
were cultivated in the absence of antibiotics for control purposes
and subjected to a routine assay using a MycoAlert Mycoplasma
Detection kit (Lonza Rockland) to ensure that they had not become
contaminated with mycoplasma.
4
9
col. For GSH quantification, the assay mixture contained 100
supernatant, 790 l of a 0.1 M sodium phosphate buffer containing
.05% EDTA at pH 7.0, 100
acid). (DTNB) dissolved in glutathione assay buffer (GAB; 125 mM
sodium phosphate containing 6.3 mM EDTA) and 10 l glutathione
reductase (55 U/ml). For GSSG quantification, the assay mixture
ll of
l
0
0
ll of 6 mM 5,5 -dithiobis(2-nitrobenzoic
l
4
.4. Cell viability assessment
comprised 100
at pH 6.8, 700
l
l of supernatant, 190
l
l of 0.5 M phosphate buffer
To determine the levels of nitrones promoting maximum and
l
l of 0.3 mM NADPH prepared in GAB, and 10
ll of
minimum cell death, concentration-dependent cytotoxicity studies
were performed. The viability of neuroblastoma cells was typically
assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
glutathione reductase (55 U/ml). The reaction rate was estimated
from the change in absorbance at 412 nm after 3 min at 25 °C
(for GSH) or at 340 nm after 16 min at 30 °C (for GSSG).
accuracy of the GSH reference standard was measured with DTNB
49–51
4
5
The
bromide (MTT) reduction assays, as previously reported. SH-
SY5Y cells were inoculated in 96-well plates at a density of
2
using a molar extinction coefficient of 13,600 with an absorbance
8
ꢂ 104 cell/cm and incubated for 24 h under the conditions
5
2
of 412 nm. GSSG was standardized by measuring the decrease
in NADPH in the presence of glutathione reductase, taking into
consideration a molar extinction coefficient of NADPH of 6270 at
described above. Aliquots of freshly prepared solutions of nitrones
18.5 mM) were added to the culture medium to attain final con-
(
centrations in the 1.0, 10.0 and 100.0
then incubated for an additional 48 h.
lM range; the plates were
3
40 nm, with 1 mol of NADPH converting 1 mol of GSSG to 2 mol
5
3
of GSH.
For cytoprotection studies, cells were preincubated with
nitrone (1.0–100 M) for 24 h and subsequently treated with
for 24 h.
l
Acknowledgments
3
2 2
00 lM H O
This work was supported by the Brazilian agencies São Paulo
Research Foundation (FAPESP, grant 2012/07717-5 and
4
.5. Apoptosis assay
2
012/01397-9) and the National Council for Scientific and
The percentage of cells undergoing apoptosis was determined
by Annexin-V staining using ApopNexin FITC Apoptosis Detection
Kit (Millipore) with a flow cytometer. SH-SY5Y cells were seeded in
TM
Technological Development (CNPq). A.C.M. is a CAPES fellow,
and G.B. is a FAPESP fellow (grant 2014/15534-3).
6
-well plates and preincubated for 24 h with PBN, 2 and 8 all at
concentrations of 1.0 M. The cells were subsequently treated with
00 M H for 24 h, harvested and washed in ice-cold PBS buf-
fer. The cell pellet was resuspended in 200 l of binding buffer
10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ) and
l
Supplementary data
3
l
2 2
O
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.12.007.
(
2
incubated with FITC-labeled Annexin-V and PI for 15 min. PI was
added to distinguish necrotic cells (Annexin-Vꢁ/PI+) from early
apoptotic cells (Annexin-V+/PIꢁ) and late apoptotic cells
References and notes
(
Annexin-V+/PI+). A flow cytometric analysis (Cytometer BD FACS-
1. Wang, Z.; Wang, Y.; Li, W.; Mao, F.; Sun, Y.; Huang, L.; Li, X. ACS Chem. Neurosci.
2014, 5, 952.
TM
Canto II) was performed to determine the percentage of apoptotic
cells in each sample.
2.
Li, Y.; Peng, P.; Tang, L.; Hu, Y.; Hu, Y.; Sheng, R. Bioorgan. Med. Chem. 2014, 22,
717.
4
3
4
5
6
.
.
.
.
Halliwell, B. Br. J. Exp. Pathol. 1989, 70, 737.
Renschler, M. F. Eur. J. Cancer 2004, 40, 1934.
4
.6. Measurement of oxidative stress
Glasauer, A.; Chandel, N. S. Biochem. Pharmacol. 2014, 92, 90.
Gupta, S. C.; Hevia, D.; Patchva, S.; Park, B.; Koh, W.; Aggarwal, B. B. Antioxid.
Redox Sign. 2012, 16, 1295.
Maccari, R.; Ottana, R. J. Med. Chem. 2015, 58, 2047.
Reuter, S.; Gupta, S. C.; Chaturvedi, M. M.; Aggarwal, B. B. Free Radical Biol. Med.
SH-SY5Y cells were plated and pre-incubated with PBN, 2, 7, 8
7
8
.
.
and 9, all at concentrations of 1.0
treated with 300 M H for 24 h. The cells were treated with a
trypsin/EDTA solution, washed three times with PBS, and resus-
pended in a 50.0 M solution of the oxidation-sensitive, nonfluo-
rescent probe 50
lM, for 24 h and subsequently
l
2 2
O
2010, 49, 1603.
9. Rajendran, P.; Rengarajan, T.; Nandakumar, N.; Palaniswami, R.; Nishigaki, Y.;
Nishigaki, I. Eur. J. Med. Chem. 2014, 86, 103.
10. Rutkowski, J. M.; Stern, J. H.; Scherer, P. E. J. Cell Biol. 2015, 208, 501.
l
l
0
0
M 2 ,7 -dichlorodihydrofluorescein diacetate
11. Wang, W. M.; Zhang, Y.; Lu, W. Y.; Liu, K. Y. Plos One 2015, 10.
4
6
(
DCFH-DA) at 37 °C for 45 min. After incubation with the probe,
12. Furukawa, S.; Fujita, T.; Shimabukuro, M.; Iwaki, M.; Yamada, Y.; Nakajima, Y.;
Nakayama, O.; Makishima, M.; Matsuda, M.; Shimomura, I. J. Clin. Invest. 2004,
the cells were washed three times with PBS, and the levels of intra-
cellular fluorescence were determined immediately by flow
1
14, 1752.

A. C. Matias et al. / Bioorg. Med. Chem. 24 (2016) 232–239
239
1
3. Tabner, B. J.; Turnbull, S.; El-Agnaf, O. M. A.; Allsop, D. Free Radical Biol. Med.
002, 32, 1076.
30. Venkatachalapathy, C.; Thirumalaikumar, M.; Muthusubramanian, S.;
Pitchumani, K.; Sivasubramanian, S. Synth. Commun. 1997, 27, 4041.
31. Vergely, C.; Renard, C.; Moreau, D.; Perrin-Sarrado, C.; Roubaud, V.; Tuccio, B.;
Rochette, L. Fundam. Clin. Pharmacol. 2003, 17, 433.
32. Chioua, M.; Sucunza, D.; Soriano, E.; Hadjipavlou-Litina, D.; Alcazar, A.; Ayuso,
I.; Oset-Gasque, M. J.; Gonzalez, M. P.; Monjas, L.; Rodriguez-Franco, M. I.;
Marco-Contelles, J.; Samadi, A. J. Med. Chem. 2012, 55, 153.
33. Woo, Y. J.; Lee, B. H.; Yeun, G. H.; Kim, H. J.; Ko, J. M.; Won, M. H.; Lee, B. H.;
Park, J. H. B. Korean Chem. Soc. 2011, 32, 2997.
34. Fang, L.; Kraus, B.; Lehmann, J.; Heilmann, J.; Zhang, Y. H.; Decker, M. Bioorgan.
Med. Chem. Lett. 2008, 18, 2905.
35. Yoo, Y. J.; Nam, D. H.; Jung, S. Y.; Jang, J. W.; Kim, H. J.; Jin, C.; Pae, A. N.; Lee, Y.
S. Bioorgan. Med. Chem. Lett. 2011, 21, 2850.
2
1
1
4. Abdelsalam, R. M.; Safar, M. M. J. Neurochem. 2015, 133, 700.
5. Moscovitz, O.; Ben-Nissan, G.; Fainer, I.; Pollack, D.; Mizrachi, L.; Sharon, M.
Nat. Commun. 2015, 6, 6609.
1
6. Fernandez-Bachiller, M. I.; Perez, C.; Gonzalez-Munoz, G. C.; Conde, S.; Lopez,
M. G.; Villarroya, M.; Garcia, A. G.; Rodriguez-Franco, M. I. J. Med. Chem. 2010,
53, 4927.
1
7. Bonda, D. J.; Wang, X. L.; Perry, G.; Nunomura, A.; Tabaton, M.; Zhu, X. W.;
Smith, M. A. Neuropharmacology 2010, 59, 290.
8. von Bernhardi, R.; Eugenin, J. Antioxid. Redox Sign. 2012, 16, 974.
9. Floyd, R. A.; Kopke, R. D.; Choi, C. H.; Foster, S. B.; Doblas, S.; Towner, R. A. Free
Radical Biol. Med. 2008, 45, 1361.
1
1
2
2
2
0. Hensley, K.; Carney, J. M.; Stewart, C. A.; Tabatabaie, T.; Pye, Q.; Floyd, R. A. Int.
Rev. Neurobiol. 1997, 40, 299.
1. Chamulitrat, W.; Parker, C. E.; Tomer, K. B.; Mason, R. P. Free Radical Res. 1995,
36. Roussaki, M.; Kontogiorgis, C. A.; Hadjipavlou-Litina, D.; Hamilakis, S.; Detsi, A.
Bioorgan. Med. Chem. Lett. 2010, 20, 3889.
37. Hinton, R. D.; Janzen, E. G. J. Org. Chem. 1992, 57, 2646.
38. Bjorgo, J.; Boyd, D. R.; Neill, D. C.; Jennings, W. B. J. Chem. Soc., Perkin Trans. 1
1977, 254.
23, 1.
2. Samadi, A.; Soriano, E.; Revuelta, J.; Valderas, C.; Chioua, M.; Garrido, I.;
Bartolome, B.; Tomassolli, I.; Ismaili, L.; Gonzalez-Lafuente, L.; Villarroya, M.;
Garcia, A. G.; Oset-Gasque, M. J.; Marco-Contelles, J. Bioorgan. Med. Chem. 2011,
39. Hwu, J. R.; Tseng, W. N.; Patel, H. V.; Wong, F. F.; Horng, D. N.; Liaw, B. R.; Lin, L.
C. J. Org. Chem. 1999, 64, 2211.
1
9, 951.
40. Mirza-Aghayan, M.; Molaee Tavana, M.; Boukherroub, R. Tetrahedron Lett.
2014, 55, 5471.
2
3. Sun, Y. W.; Zhang, G. X.; Zhang, Z. J.; Yu, P.; Zhong, H. J.; Du, J.; Wang, Y. Q.
Bioorgan. Med. Chem. 2012, 20, 3939.
4. Costa, D. S. S.; Martino, T.; Magalhaes, F. C.; Justo, G.; Coelho, M. G. P.; Barcellos,
J. C. F.; Moura, V. B.; Costa, P. R. R.; Sabino, K. C. C.; Dias, A. G. Bioorgan. Med.
Chem. 2015, 23, 2053.
41. Esat, B.; Fidan, I.; Bahceci, S.; Yerli, Y.; Sari, L. Magn. Reson. Chem. 2009, 47, 641.
42. Nelson, D. W.; Owens, J.; Hiraldo, D. J. Org. Chem. 2001, 66, 2572.
43. Wang, L.; Xie, X.; Liu, Y. H. Org. Lett. 2012, 14, 5848.
44. Xiao, Z. M.; Huang, C.; Wu, J.; Sun, L.; Hao, W.; Leung, L. K.; Huang, J. Eur. J.
Pharmacol. 2013, 721, 286.
2
2
2
5. Das, A.; Gopalakrishnan, B.; Voss, O. H.; Doseff, A. I.; Villamena, F. A. Biochem.
Pharmacol. 2012, 84, 486.
45. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
6. Samadi, A.; Soriano, E.; Revuelta, J.; Valderas, C.; Chioua, M.; Garrido, I.;
Bartolome, B.; Tomassolli, I.; Ismaili, L.; Gonzalez-Lafuente, L.; Villarroya, M.;
Garcia, A. G.; Oset-Gasque, M. J.; Marco-Contelles, J. Bioorgan. Med. Chem. 2011,
46. LeBel, C. P.; Ischiropoulos, H.; Bondy, S. C. Chem. Res. Toxicol. 1992, 5, 227.
47. Filomeni, G.; Cerchiaro, G.; Ferreira, A. M. D.; De Martino, A.; Pedersen, J. Z.;
Rotilio, G.; Ciriolo, M. R. J. Biol. Chem. 2007, 282, 12010.
1
9, 951.
48. Behl, C.; Davis, J. B.; Lesley, R.; Schubert, D. Cell 1994, 77, 817.
49. Hafer, K.; Iwamoto, K. S.; Schiestl, R. H. Radiat. Res. 2008, 169, 460.
50. Abilio, V. C.; Araujo, C. C.; Bergamo, M.; Calvente, P. R.; D’Almeida, V.; Ribeiro
Rde, A.; Frussa-Filho, R. Prog. Neuropsychopharmacol. Biol. Psychiatr. 2003, 27,
109.
2
2
7. Arce, C.; Diaz-Castroverde, S.; Canales, M. J.; Marco-Contelles, J.; Samadi, A.;
Oset-Gasque, M. J.; Gonzalez, M. P. Eur. J. Med. Chem. 2012, 55, 475.
8. Rao, P. S.; Kurumurthy, C.; Veeraswamy, B.; Kumar, G. S.; Poornachandra, Y.;
Kumar, C. G.; Vasamsetti, S. B.; Kotamraju, S.; Narsaiah, B. Eur. J. Med. Chem.
2
014, 80, 184.
51. Tietze, F. Anal. Biochem. 1969, 27, 502.
2
9. Chavarria, C.; Perez, D. I.; Perez, C.; Morales Garcia, J. A.; Alonso-Gil, S.; Perez-
52. Eyer, P.; Podhradsky, D. Anal. Biochem. 1986, 153, 57.
53. Akerboom, T. P.; Sies, H. Methods Enzymol. 1981, 77, 373.
Castillo, A.; Gil, C.; Souza, J. M.; Porcal, W. Eur. J. Med. Chem. 2012, 58, 44.